Journal: Frontiers in Microbiology

Article Title: Human beta defensin-3 mediated activation of β-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5

doi: 10.3389/fmicb.2023.1186510

Figure Lengend Snippet: Human defensin-3 induces β-catenin activity in lung epithelial cells. (A) Human lung epithelial A549 cells were treated with either vehicle (0.1% BSA in PBS) or purified human defensin-3 (HBD3) protein (10 μg/ml) for 16 h. β-catenin and actin levels were determined in cell lysates by western blotting using corresponding antibodies. (B) Densitometry analysis of β-catenin protein levels relative to actin protein (β-catenin/Actin) in the vehicle and HBD3-treated A549 cells. (C) TOP-Flash luciferase assay of A549 cells co-transfected with firefly-luciferase-TOP-Flash and renilla-luciferase plasmids. Co-transfected cells were treated with either vehicle (0.1% BSA in PBS) or HBD3 for 16 h. A dual luciferase reagent was utilized to determine firefly and renilla luciferase activity. The relative TOP-Flash luciferase activity was calculated based on the mean value of firefly/renilla luciferase activity. The value is represented as a fold change in TOP-Flash activity in HBD3-treated cells compared to vehicle-treated cells. The densitometric values represent the mean ± SEM from three independent studies (* p ≤ 0.05). Luciferase assay represents mean ± SEM from two independent experiments performed in triplicates [* p ≤ 0.05 ( n = 6; technical replicates)].

Article Snippet: HBD3-8A antibody was deposited to the DSHB by Starner, T. (DSHB Hybridoma Product hBD-3-8A).

Techniques: Activity Assay, Purification, Western Blot, Luciferase, Transfection

Journal: Frontiers in Microbiology

Article Title: Human beta defensin-3 mediated activation of β-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5

doi: 10.3389/fmicb.2023.1186510

Figure Lengend Snippet: RSV induces HBD3 expression and release from lung epithelial cells. (A) Human lung epithelial A549 cells were infected with RSV (MOI = 1) for 16 h. RNA isolated from these cells was subjected to RT-PCR to analyze the expression of HBD3 and GAPDH (loading control). The PCR from three independent experiments (each lane corresponds to each independent experiment) are shown in the mock-infected and RSV-infected panels. (B) Densitometry analysis of HBD3 mRNA levels relative to GAPDH mRNA (HBD3/GAPDH) in mock vs. RSV-infected (16 post-infection) A549 cells. (C) Medium supernatant collected from RSV-infected (MOI = 1; 16 h post-infection) A549 cells were analyzed for HBD3 release by ELISA. The densitometric values represent the mean ± SEM from three independent studies (* p ≤ 0.05). ELISA data are shown as Mean ± SEM [* p ≤ 0.05 ( n = 18; technical replicates; three independent experiments)].

Article Snippet: HBD3-8A antibody was deposited to the DSHB by Starner, T. (DSHB Hybridoma Product hBD-3-8A).

Techniques: Expressing, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: Human beta defensin-3 mediated activation of β-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5

doi: 10.3389/fmicb.2023.1186510

Figure Lengend Snippet: HBD3 promotes β-catenin protein stabilization during RSV infection of lung epithelial cells. (A) Human lung epithelial A549 cells were transfected with either control scrambled siRNA or siRNA specific for HBD3. HBD3 and actin levels were determined in cell lysates of siRNA-transfected cells by western blotting using corresponding antibodies. (B) Densitometry analysis of HBD3 protein levels relative to actin protein (HBD3/Actin) in control siRNA and HBD3 siRNA transfected A549 cells. (C) Control siRNA and HBD3 siRNA transfected cells were infected with RSV (MOI = 1) for 16 h. β-catenin and actin levels were determined in cell lysates by western blotting using corresponding antibodies. (D) Densitometry analysis of β-catenin protein levels relative to actin protein (β-catenin/Actin) in RSV-infected A549 cells transfected with either control siRNA or HBD3 siRNA. The densitometric values represent the mean ± SEM from three independent studies (* p ≤ 0.05).

Article Snippet: HBD3-8A antibody was deposited to the DSHB by Starner, T. (DSHB Hybridoma Product hBD-3-8A).

Techniques: Infection, Transfection, Western Blot

Journal: Frontiers in Microbiology

Article Title: Human beta defensin-3 mediated activation of β-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5

doi: 10.3389/fmicb.2023.1186510

Figure Lengend Snippet: Interaction of HBD3 with LRP5. (A) Human lung epithelial A549 cells were transfected with either empty FLAG plasmid (FLAG-empty) or plasmid encoding FLAG-tagged LRP5 protein (FLAG-LRP5). Chilled FLAG-empty and FLAG-LRP5 A549 cells were incubated with biotinylated purified HBD3 protein (Biotin-HBD3) at 4°C for 4 h. Following incubation, cell lysates were precipitated (PPT) with avidin-agarose and subsequently immuno-blotted (IB) with FLAG antibody to detect LRP5. Whole-cell lysates (WCL) were also blotted with FLAG and actin antibodies. (B) A schematic showing the domain structure of the LRP5 protein. (C) Biot-HBD3 and non-biotinylated HBD3 (control) were precipitated (PPT) with avidin-agarose. The avidin-agarose was then incubated with purified truncated LRP5 protein comprising of the E3 and E4 domains of the extracellular domain of LRP5. Following incubation, the agarose-avidin-bound protein was subjected to immunoblotting with the LRP5 antibody. The immune blots are representative of three independent experiments with similar results.

Article Snippet: HBD3-8A antibody was deposited to the DSHB by Starner, T. (DSHB Hybridoma Product hBD-3-8A).

Techniques: Transfection, Plasmid Preparation, Incubation, Purification, Avidin-Biotin Assay, Western Blot

Journal: Frontiers in Microbiology

Article Title: Human beta defensin-3 mediated activation of β-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5

doi: 10.3389/fmicb.2023.1186510

Figure Lengend Snippet: The plausible binding modes and critical interactions of the binary (LRP5-HBD3) and ternary (LRP5-HBD3-FZD8) complexes. (A,B) Protein–protein docking and molecular dynamics simulations revealed potential binding modes of the LRP5-HBD3 and LRP5-HBD3-FZD8 interaction complexes, respectively. The residues involved in the formation of the complex are shown in surface representation, and LPR5, HBD3, and FZD8 are colored in steel blue, light pink, and teal, respectively. HBD3 binds between the β-propeller domain-3 of LPR5 and the cysteine-rich domain of the FZD8 receptor. FZD8 receptor interacts with HBD3 as well as with the LRP5 receptor residues. (C) Stable H-bond interactions observed during the MD simulations of the complex are highlighted in boxes. (D) The sidechain carboxylic group of E714 of LRP5 interacts with the backbone amide nitrogen of M1 of HBD3, and the backbone amide nitrogen of H974 of LRP5 interacts with side chain ε nitrogen of H4 of HBD3. (E) The sidechain amino group of K675 of LRP5 interacts with backbone carbonyl oxygens of L13 and L15 of HBD3. (F) The sidechain amino group of K30 of HBD3 interacts with the ε nitrogen of FZD8 H124. Similarly, the backbone amide nitrogen of C40 of HBD3 forms an H-bond with the backbone carbonyl oxygen of FZD8 G20. (G) The sidechain terminal amino group of K953 of LRP5 forms a salt bridge with the sidechain carboxyl group of E50. Similarly, the sidechain amino group of K697 of LRP5 formed an H-bond with the backbone carbonyl oxygen of E50 of the FZD8 receptor.

Article Snippet: HBD3-8A antibody was deposited to the DSHB by Starner, T. (DSHB Hybridoma Product hBD-3-8A).

Techniques: Binding Assay

Journal: Frontiers in Microbiology

Article Title: Human beta defensin-3 mediated activation of β-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5

doi: 10.3389/fmicb.2023.1186510

Figure Lengend Snippet: Critical H-bond interactions and the extent of protein–protein interactions as measured by the buried solvent-accessible surface area (SASA) of individual proteins upon complex formation. (A) Multiple stable H-bond interactions were observed in the LRP5-HBD3-FZD8 ternary complex. H-bond interactions between LRP5-HBD3, HBD3-FZD8, and LRP5-FZD8 are shown. (B) Total solvent-accessible surface area (SASA) of the three proteins in their unbound states and the amount of SASA lost due to complex formation, called buried surface area (BSA) and %, are given. (C) The total BSA and the fraction of the buried surface area for each interacting partner of HBD3, LRP5, and FZD8 receptors were calculated from the MD simulation. The BSA of HBD3 shows that HBD3 shares its interaction surface approximately equally with LRP5 and FZD8 receptors. On the other hand, LRP5 and FZD receptors share a larger fraction of interaction surface with HBD3. The fraction of interaction surface for LRP5 and FZD8 receptors was relatively smaller in comparison to their total SASA.

Article Snippet: HBD3-8A antibody was deposited to the DSHB by Starner, T. (DSHB Hybridoma Product hBD-3-8A).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Human beta defensin-3 mediated activation of β-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5

doi: 10.3389/fmicb.2023.1186510

Figure Lengend Snippet: Schematic diagram of the proposed model for LRP5-HBD3-FZD8 ternary complex. Protein–protein docking, and MD simulations predicted the potential interactions of HBD3 with LRP5 and FZD8. HBD3 appears to be sandwiched between LRP5 and FZD8. Specifically, HBD3 binds mostly to the PE3 propeller domain of LRP5 protein.

Article Snippet: HBD3-8A antibody was deposited to the DSHB by Starner, T. (DSHB Hybridoma Product hBD-3-8A).

Techniques:

Journal: European Journal of Histochemistry : EJH

Article Title: Histochemical localization of sialic acids and antimicrobial substances in eccrine glands of porcine snout skin

doi: 10.4081/ejh.2012.e6

Figure Lengend Snippet: mmunohistochemical staining in the eccrine glands of the snout skin. a) Lysozyme, arrow: dark cell; b) IgA, C: capillary, arrow: dark cell; c) lactoferrin, arrow: dark cell; d) β-defensin 2, arrow: dark cell; e) Rab3D, arrow: dark cell; f) control.

Article Snippet: Then, they were incubated with primary antibodies diluted in 0.01 M PBS (pH 7.3) against lysozyme (polyclonal, from rabbit) (Dako, Glostrup, Denmark), IgA (polyclonal, from rabbit) (Dako), lactoferrin (polyclonal, from rabbit) (MP Biomedicals, Ohio, USA), human β-defensin 2 (polyclonal, from rabbit) (Biolog, Kronshagen, Germany) and Rab3D (polyclonal, from rabbit) (Proteintech, Illinois, USA) for 24 h at 4°C, following preincubation with 1% BSA in PBS for 30 min at room temperature.

Techniques: Staining

Journal: The Journal of Investigative Dermatology

Article Title: IL-1β–Induced Protection of Keratinocytes against Staphylococcus aureus -Secreted Proteases Is Mediated by Human β-Defensin 2

doi: 10.1016/j.jid.2016.08.025

Figure Lengend Snippet: IL-1β treatment induces the expression of hBD2 in keratinocytes, a soluble factor that protects against V8 protease-mediated damage. ( a ) HaCaT (n = 9) or HPEK (n = 3) cells treated with conditioned media (transferred from control or IL-1β [100 ng/ml] pretreated cells) were exposed to recombinant V8 protease for 24 hours before quantification of damage. ( b–f ) HaCaT or HPEK cells treated for 24 hours or ( e ) over a time course with control (media only), IL-1β (100 ng/ml), LTA (10 μg/ml), LPS (5 μg/ml), or heat-killed Staphylococcus epidermidis, then assessed by ( b ) real-time reverse transcriptase PCR for expression of DEFB4 , DEFB1 , ( c, e, f ) ELISA for hBD2, and ( d ) quantification of V8-induced damage. Data show mean ± standard error of the mean. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus control. CM, conditioned media; HKSE, heat-killed S. epidermidis ; HPEK, human primary epidermal keratinocyte; LPS, lipopolysaccharide; LTA, lipoteichoic acid; NS, not significant.

Article Snippet: Standard curves were performed using plasmids for DEFB1 , DEFB4 (Origene #SC116851, RC219487), and DEFB103 (generated by J. Dorin).

Techniques: Expressing, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Investigative Dermatology

Article Title: IL-1β–Induced Protection of Keratinocytes against Staphylococcus aureus -Secreted Proteases Is Mediated by Human β-Defensin 2

doi: 10.1016/j.jid.2016.08.025

Figure Lengend Snippet: hBD2 is sufficient and necessary to protect against V8 protease-mediated damage. ( a–e ) hBD2 overexpressing (hBD2 OE) and vector only (VO control) HaCaT or naive HaCaT preincubated with CM or ( f, g ) DEFB4 shRNA-knockdown and control shRNA HaCaT lines ± IL-1β prestimulation (100 ng/ml, 24 hours), assessed by ( a ) DEFB4 quantitative real-time reverse transcriptase–PCR (n = 3), ( b, f ) hBD2 ELISA (n = 5–7), ( c, d, g ) V8-mediated damage quantification (n = 3–4), and ( e ) quantified Western blot ± V8 pre-exposure (3 μg/ml, 24 hours). ( h, i ) HaCaT or HPEK pretreated with distilled water, recombinant (3 μg/ml), scrambled ( h : 3 μg/ml, i : 6 μg/ml), or synthetic hBD2 ( h : 3 μg/ml, i : 6 μg/ml ) 24 hours before V8 ( h : 24 hours, 3 μg/ml; i : 48 hours, 6 μg/ml). n = 3–5. Mean ± standard error of the mean. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. CM, conditioned media; hBD, human β-defensin; HPEK, human primary epidermal keratinocyte; OE, overexpressing; Recomb., recombinant; Scramb., scrambled; shRNA, small hairpin RNA; Synth, synthetic; VO, vector only.

Article Snippet: Standard curves were performed using plasmids for DEFB1 , DEFB4 (Origene #SC116851, RC219487), and DEFB103 (generated by J. Dorin).

Techniques: Plasmid Preparation, shRNA, Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant

Journal: Placenta

Article Title: SWATH proteomics analysis of placental tissue with intrahepatic cholestasis of pregnancy.

doi: 10.1016/j.placenta.2023.04.009

Figure Lengend Snippet: Fig. 7. Western blot analysis of differentially expressed proteins in placental tissue of ICP patients and controls. A) Western blot of PDE3A, HPX, PRG4, HBD, and β-ACTIN. B) The relative protein expression (n = 3). Each bar representing the standard deviation (SD; P < 0.05). The image analyzed by Western blot analysis on the left corresponds to the semi-quantitative histogram on the right. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The membranes were closed with 5% milk for 1 h at room temperature, washed and incubated overnight at 4 ◦C with the following primary antibodies: anti-HBD antibody (1:2000 dilution; Proteintech Group; No: 16824-1-AP), anti-HPX antibody (1:3000 dilution; Proteintech Group; No: 15736-1-AP), anti-PDE3A antibody (1:30000 dilution; Affinity Biosciences; No: DF9371), anti-PRG4 antibody (1:2000 dilution; Proteintech Group; No: 25732-1-AP), and anti-β-ACTIN antibody (1:10000 dilution; Proteintech Group; No: 81115-1-RR).

Techniques: Western Blot, Expressing, Standard Deviation